Novel Dosing Regimens of Celgosivir for the Treatment of Dengue

ABSTRACT

Methods of treating a dengue virus (DENV) infection in a human subject, comprising administering to the human subject a compound of Formula (I), or pharmaceutical composition comprising a compound of Formula (I): A compound of Formula (I) can be administered within onset of fever to 72 hours of fever onset due to dengue infection and then every 6 to 12 hours until there is an improvement in the infection or between from about 1 day to about 10 days. The methods of the invention can be used to treat primary and secondary DENV1-4 viral infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/798,100, filed on Mar. 15, 2013, and U.S. ProvisionalPatent Application No. 61/911,795, filed on Dec. 4, 2013, the entiretiesof which are incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

Globally, dengue infections result in more than 20,000 deaths, nearly500,000 hospitalized cases and anywhere between 50-100 million humaninfections annually. (1) Dengue infection is caused by one of fourimmunologically distinct serotypes of the dengue virus (DENV 1-4). Thevirus is spread by the urban breeding mosquito Aedes aegypti. Usually,infection with any one of the four DENV serotypes leads to mild,self-limiting dengue fever with lifelong immunity to the specificserotype of infection. Epidemiological evidence also indicates that 90%of the severe and potentially fatal dengue diseases, dengue hemorrhagicfever (DHF) or dengue shock syndrome (DSS) occur during secondaryheterotypic infections where the protective antibody from a priorinfection takes on a pathogenic role, so-called Antibody DependentEnhancement (ADE). (2, 3) The antibody response triggers a systemicinflammatory reaction resulting in vascular leakage.

Currently, there is no approved preventive vaccine or antiviraltreatment for dengue disease. The World Health Organization has listeddengue fever as an emerging and uncontrolled disease. (4) Hence, thereis an urgent medical need for the development of a potent dengueantiviral that is safe for use in humans.

SUMMARY OF THE INVENTION

The present invention pertains to methods of treating a dengue virus(DENV) infection in a human subject. In one aspect, the method comprisesadministering to the human subject an initial (loading) dose of about100 to 600 mg of a compound of Formula (I), or a pharmaceuticalcomposition comprising a compound of Formula (I), within from aboutonset of fever to about 72 hours of fever onset due to dengue infection,followed by administration of One or more subsequent doses of about 25to about 300 mg of a compound of Formula (I), or a pharmaceuticalcomposition comprising a compound of Formula (I).

In one embodiment, the subsequent doses are administered at intervals offrom about 6 to about 12 hours. In other embodiments, the subsequentdoses are administered about every 6 hours, about every 8 hours, aboutevery 12 hours, or about every 24 hours.

In certain embodiments, the subsequent doses are administered for about1-10 days. In other embodiments, the subsequent doses are administeredfor about 1-5 days, 1-4 days, 1-3 days, 2-3 days, or 1-2 days. Infurther embodiments, the subsequent doses are administered until thereis an improvement in the infection or its symptoms.

In the various embodiments, the human subject can be an adult or achild.

The compound of Formula (I) is represented by the following structure:

or a pharmaceutically acceptable salt thereof:

wherein R¹, R², and R³ are independently H, (C₁-C₁₄) acyl, (C₁-C₁₄)alkenylacyl, (C₃-C₈) cycloalkylacyl, (C₁-C₁₄) haloalkylacyl (C₁-C₈)alkoxyacyl, or (C₆-C₁₀) arylacyl.

In certain embodiments, the compound of Formula (I) is specifically thecompound of Formula (II), below, or a pharmaceutically acceptable saltthereof:

In preferred embodiments, the compound of Formula (I) is prodrug ofcastanospermine, a natural product derived from the seeds ofCastanospermum australe. Once administered compounds of Formula (I) arerapidly converted to castanospermine. Compounds of Formula (I) (e.g.,celgosivir) are more rapidly and efficiently absorbed thancastanospermine. Compounds of Formula (I) are also more readily absorbedinto cells. As a result, compounds of Formula (I) may have higher 50%effective concentration (EC50) values and in vivo efficacy thancastanospermine against the dengue (DENV) virus.

In certain embodiments, the initial dose is the same as the subsequentdoses, while in other embodiments the initial dose differs from thesubsequent doses. In particular embodiments, the initial dose is higherthan the subsequent doses.

In certain embodiments, for an adult subject, the initial dose of acompound of Formula (I) can be between about 100 to about 600 mg. Inother embodiments, the initial dose of a compound of Formula (I) in anadult subject can be about 150-600 mg, about 200-500 mg, or about250-400 mg. In further embodiments, the initial dose of a compound ofFormula (I) in an adult subject can be about 100 mg, about 125 mg, about150 mg, about 175 mg about 200 mg, about 225 mg, about 250 mg, about 275mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525mg, about 550 mg, about 575 mg, or about 600 mg. In a furtherembodiment, the initial dose of a compound of Formula (T) in an adultsubject is between about 550 to about 600 mg. In another embodiment, theinitial dose of a compound of Formula (I) in an adult subject is betweenabout 500 to about 550 mg. In yet another embodiment, the initial doseof a compound of Formula (I) in an adult subject is between about 450 toabout 500 mg. In further embodiment, the initial dose of a compound ofFormula (I) in an adult subject is between about 400 to about 450 mg. Inanother embodiment, the initial dose of a compound of Formula (I) in anadult subject is between about 350 to about 400 mg. In furtherembodiment, the initial dose of compound of Formula (I) in an adultsubject is between about 300 to about 350 mg. In yet another embodiment,the initial dose of a compound of Formula (I) in an adult subject isbetween about 250 to about 300 mg. In further embodiment, the initialdose of compound of Formula (I) in an adult subject is between about 200to about 250 mg. In another embodiment, the initial dose of a compoundof Formula (I) in an adult subject is between about 150 to about 200 mg.In another embodiment, the initial dose of compound of Formula (I) in anadult subject is between about 100 to about 150 mg.

The subsequent doses of a compound of Formula (I) in an adult subjectcan be between about 100 to about 300 mg. In one embodiment, thesubsequent dose of a compound of Formula (I) in an adult subject isbetween about 250 to about 300 mg. In another embodiment the subsequentdose of a compound of Formula (I) in an adult subject is between about200 to about 250 mg. In yet another embodiment, the subsequent dose of acompound of Formula (I) in an adult subject is between about 150 toabout 200 mg. In further embodiment, the subsequent dose of a compoundof Formula (I) in an adult subject is between about 100 to about 200 mg.In an additional embodiment, the subsequent dose of a compound ofFormula (I) in an adult subject is between about 125 to about 175 mg. Inyet another embodiment, the subsequent dose of a compound of Formula (I)in an adult subject is about 150 mg.

For a child subject, the initial dose of a compound of Formula (I) in achild subject can be between about 25 to about 450 mg. In oneembodiment, the initial dose of a compound of Formula (I) in a childsubject is between about 25 to about 50 mg. In another embodiment, theinitial dose of a compound of Formula (I) in a child subject is betweenabout 50 to about 75 mg. In yet another embodiment, the initial dose ofa compound of Formula (I) in a child subject is between about 75 toabout 100 mg. In another embodiment, the initial dose of a compound ofFormula (I) in a child subject is between about 100 to about 150 Mg. Inanother embodiment, the initial dose of a compound of Formula (I) in achild subject is between about 150 to about 200 mg. In yet anotherembodiment, the initial dose of a compound of Formula (I) in a childsubject is between about 200 to about 250 mg. In further embodiment, theinitial dose of a compound of Formula (I) in a child subject is betweenabout 250 to about 300 mg. In another embodiment, the initial (lose of acompound of Formula (I) in a child subject is between about 300 to about350 mg. In yet another embodiment, the initial dose of a compound ofFormula (I) in a child subject is between about 350 to about 400 mg.

The subsequent doses of a compound of Formula (I) in a child subject canbe between about 25 to about 200 mg. In one embodiment, the subsequentdose of a compound of Formula (I) in a child subject is between about 25to about 50 mg, in another embodiment, the subsequent dose of a compoundof Formula (I) in a child subject is between about 50 to about 75 mg. Inyet another embodiment, the subsequent dose of a compound of Formula (I)in a child subject is between about 75 to about 100 mg. In furtherembodiment, the subsequent dose of a compound of Formula (I) in a childsubject is between about 100 to about 125 mg. In another embodiment, thesubsequent dose of a compound of Formula (I) in a child subject isbetween about 125 to about 150 mg. In yet another embodiment, thesubsequent dose of a compound of Formula (I) in a child subject isbetween about 150 to about 200 mg.

In one embodiment, the initial dose is administered at the time of leveronset due to dengue infection. In another embodiment, the initial doseis administered within 24 hours of fever onset due to dengue infection.In yet another embodiment, the initial dose is administered within 48hours of fever onset due to dengue infection. In a further anotherembodiment, the initial dose is administered within 72 hours of feveronset due to dengue infection.

The compounds or pharmaceutical compositions of the present inventioncan be administered intravenously, orally, rectally or sublingually. Inone embodiment, the route of administration is intravenous. In anotherembodiment, the route of administration is oral. In another embodiment,the route of administration is rectal. In yet another embodiment, theroute of administration is sublingual.

The compounds or pharmaceutical compositions of the present inventioncan be administered as a single or as a divided dose. Preferably, theinitial dose is a single dose. In some embodiments, the subsequent dosescan be single, divided, or a combination thereof, during the course oftherapy depending upon patient and progress of the infection. For thesubsequent doses in one embodiment, the human subject is administered adivided dose of from about 25 to about 300 mg of a compound of Formula(I), or a pharmaceutical composition comprising a compound of Formula(I), for between about 1 day to about 10 days. In another embodiment,the human subject is administered a single dose of from about 25 toabout 300 mg of a compound of Formula (I), or a pharmaceuticalcomposition comprising a compound of Formula (I), for between about 1day to about 10 days. In yet another embodiment, the human subject isadministered a single dose of from about 25 to about 300 mg of acompound of Formula (I), or a pharmaceutical composition comprising acompound of Formula (I), for between about 1 day to about 2 days. In afurther embodiment, the human subject is administered a single dose offrom about 25 to about 300 mg of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), forbetween about 2 days to about 5 days. In other versions, the subsequentdoses are administered no longer than about 1 day; in yet other versionsno longer than about 2 days; in further versions no longer than about 5days; and other versions no longer than about 10 days.

The invention also relates to methods of treating a dengue virusinfection by achieving a steady state Cmin serum or plasma concentrationof between about 0.05 and about 2.0 microgram/mL of castanospermine inan adult or child subject. In one embodiment, the steady state Cminserum or plasma concentration achieved in an adult or child subject isbetween about 0.08 and about. 0.5 microgram/mL of castanospermine. Inanother embodiment, the steady state Cmin serum or plasma concentrationachieved in an adult or child subject is between about 0.05 and about0.08 microgram/mL of castanospermine. In yet another embodiment, thesteady state Cmin serum or plasma concentration achieved in an adult orchild subject is between about 0.08 and about 0.11 microgram/mt, ofcastanospermine. In a further embodiment, the steady state Cmin serum orplasma concentration achieved in an adult or child subject is betweenabout 0.11 and about 0.3 microgram/mL of castanospermine. In anotherembodiment, the steady state Cmin serum or plasma concentration achievedin an adult or child subject is between about 0.3 and about 0.75microgram/mL of castanospermine. In further embodiment, the steady stateCmin serum or plasma concentration achieved in an adult or child subjectis between about 0.75 and about 1.0 microgram/mL of castanospermine. Inyet another embodiment, the steady state Cmin serum or plasmaconcentration achieved in an adult or child subject is between about 1.0and about 2.0 microgram/mL of castanospermine. In another embodiment,the steady state Cmin serum or plasma concentration achieved in an adultor child subject is between about 1.0 and about 1.5 microgram/mL ofcastanospermine. In further embodiment, the steady state Cmin serum orplasma concentration achieved in an adult or child subject is betweenabout 1.5 and about 2.0 microgram/mL of castanospermine. In yet anotherembodiment, the steady state Cmin serum or plasma concentration achievedin an adult or child subject is between about 1.25 and about 1.75microgram/mL, of castanospermine.

In another aspect, the invention relates to methods for treating adengue viral infection comprising at least one dengue virus selectedfrom DENV1, DENV2, DENV3 and DENV4. In one embodiment, the dengue virusis DENV1. In another embodiment, the dengue virus is DENV2. In yetanother embodiment, the dengue virus is DENV3. In .further embodiment,the dengue virus is DENV4.

In yet another aspect, the invention relates to methods of treating adengue viral infection in a human subject who has tested positive fordengue virus. Known methods for diagnosis of dengue viral infection canbe used including, but are not limited to, an NS1 (nonstructuralprotein 1) strip assay or a quantitative Polymerase Chain Reaction (PCR)assay. The selected method should be rapid enough for a diagnosis withinfrom about onset of fever to about 72 hours of fever onset to optimizethe therapeutic regimen of the various embodiments of the invention.

The invention also relates to methods of treating a secondary dengue(DENV) viral infection in a human subject, comprising administering tothe human subject an initial dose of about 100 to about 600 mg of acompound of Formula (I), or a pharmaceutical composition comprising acompound of Formula (I), within from about onset of lever to about 72hours of fever onset due to dengue infection and administering to thehuman subject a dose of about 25 to about 300 mg of a compound ofFormula (I), or a pharmaceutical composition comprising a compound ofFormula (I), at intervals of from about 6 to about 12 hours until thereis an improvement in the infection or between from about 1 day to about10 days.

In another aspect of the invention, viral load reduction of treatedhuman subjects is at least 50% greater than untreated or placebo-treatedhuman subjects. In one embodiment, the virological log reduction inhuman subjects treated with a compound of Formula (I) is at least 50%greater than untreated or placebo-treated groups. In another embodiment,the virological log reduction in human subjects treated with a compoundof Formula (I) is between about 60 to about 70% greater than untreatedor placebo-treated groups. In another embodiment, the virological logreduction in human subjects treated with a compound of Formula (I) isbetween about 70 to about 80% greater than untreated or placebo-treatedgroups. In yet another embodiment, the virological log reduction inhuman subjects treated with a compound of Formula (I) is between about80 to about 90% greater than untreated or placebo-treated groups.

The invention is also directed to methods of treating a dengue viralinfection comprising administering a pharmaceutical compositioncomprising a compound of Formula (I), Formula (II), or Formula (III)according to any one of the dosing regimens described herein. Thedisclosed compounds of Formula (I), Formula (TT) or Formula (III) cartbe administered to the subject in conjunction with an acceptablepharmaceutical carrier or diluent as part of a pharmaceuticalcomposition for treatment of a dengue (DENV) infection.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings.

FIG. 1 shows a concentration response curve of celgosivir on DENV2infection in the Cell-based flaviviral immunodetection assay (CFI)assay; EC50 ranges from 0.2 to 0.7 μM.

FIG. 2 shows immunofluorescence microscopy of untreated and celgosivir(20 μM) treated DENV2 infected BHK21. Nuclei are stained with bluefluorescent DAPI. Virus replication was detected using monoclonalanti-NS1 antibody (green fluorescence), The top row shows thatuntreated, infected cells have abundant NS1 in the cytoplasm while thebottom row shows that infected cells treated with celgosivir have verylow levels of NS1. Low levels of NS1 indicate suppression of viralreplication.

FIG. 3 shows the concentration response curve of celgosivir on DENV2infection in the replicon assay (EC₅₀=2.2 μM).

FIG. 4A shows the ADE effect—antibody against dengue E protein increasesviremia in THP-1 human monocytes infected with dengue virus. FIG. 4Bshows the effect of celgosivir on DENV2 infected human monocytespretreated with antibody to DENV E protein.

FIG. 5A shows a schematic of the dosing conducted in the DENV2 infectedmice in a lethal ADE model of viremia. FIG. 5B shows celgosivir improvessurvival of DENV2 infected mice in a lethal ADE model of viremia in adose- and schedule-dependent manner. Survival at day 12 was 1/8 (13%) at10 mg/kg twice daily (BID), 5/8 (63%) at 25 mg/kg BID, 7/7 (100%) at 50mg/kg BID, and 0/8 (0%) at 100 mg/kg once daily (QD).

FIG. 6A shows a concentration profile of celgosivir and castanospermineafter a single 50 mg/kg IP dose. FIG. 6B shows a castanospermine profilecalculated for 50 mg/kg BID dosing regimen.

FIG. 7 shows the castanospermine concentration-time data over thetreatment period. Circles represent observed concentrations. Shaded greyarea represents the model predicted 10^(th) to 90^(th) predictioninterval. Solid line represents the 50^(th) prediction interval.

FIG. 8A shows a simulated population mean castanospermine, exposureafter a 400 mg loading dose of celgosivir followed by 9 subsequent dosesof 200 mg of celgosivir given at 12 hour intervals. 8B shows a simulatedpopulation mean castanospermine exposure after a 400 mg loading dose ofcelgosivir followed by 4 subsequent closes of 200 mg of celgosivir givenat 12 hour intervals. 8C shows a simulated population meancastanospermine exposure after a 400 mg loading dose of celgosivirfollowed by 8 subsequent doses of 100 mg of celgosivir given at 6 hourintervals. 8D shows a simulated population mean castanospermine exposureafter a 400 mg loading close of celgosivir followed by 6 subsequentdoses of 133 mg of celgosivir given at 8 hour intervals. 8E shows asimulated population mean castanospermine exposure after a 300 mgloading dose of celgosivir followed by 3 subsequent doses of 300 mg ofcelgosivir given at 12 hour intervals. 8F shows a simulated populationmean castanospermine exposure after a 200 mg loading dose of celgosivirfollowed by 5 subsequent doses of 200 mg of celgosivir given at 8 hourintervals. 8G shows a simulated population mean castanospermine exposureafter a 150 mg loading dose of celgosivir followed by 7 subsequent dosesof 150 mg of celgosivir given at 6 hour intervals. 8H shows a simulatedpopulation mean castanospermine exposure after a single dose of 600 mg.For FIGS. 8A-8H the solid line represents the simulated dosing regimen,the dotted line represents a dosing regimen of 400 mg initial closefollowed by 200 mg every 12 hours for 9 doses (i.e. 5 days of treatment)and the dashed line represents target minimum trough concentration. LDis loading dose, q is ‘every’ and refers to the duration of time betweendoses.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Definitions

All definitions of substituents set forth below are further applicableto the use of the term in conjunction with another substituent. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

“Alkyl” as used alone or as part of a larger moiety as in “arylalkyl” or“aryloxyalkyl” means a saturated aliphatic branched or straight-chainmonovalent hydrocarbon radicals, typically C1-C16, preferably C1-C12,For example, “(C1-C6) alkyl” means a radical having from 1-6 carbonatoms in a linear or branched arrangement, “(C1-C6) alkyl” includesmethyl, ethyl, propyl, butyl, tert-butyl, pentyl and hexyl.

“Alkylene” means a saturated aliphatic straight-chain divalenthydrocarbon radical. Thus, “(C₁-C₆) alkylene” means a divalent saturatedaliphatic radical having from 1-6 carbon atoms in a linear arrangement.“(C₁-C₆) alkylene” includes methylene, ethylene, propylene, butylene,pentylene and hexylene.

“Cycloalkyl” means saturated aliphatic cyclic hydrocarbon ring. Thus,“C₃-C₈ cycloalkyl” means (3-8 membered) saturated aliphatic cyclichydrocarbon ring. C₃-C₈ cycloalkyl includes, but is not limited tocyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. Preferably, cycloalkyl is C₃-C₆ cycloalkyl.

The term “alkoxy” means —O-alkyl; “arylalkoxy” means an alkoxy groupsubstituted at any carbon by an aryl group; “hydroxyalkyl” means alkylsubstituted with hydroxy; “arylalkyl” means alkyl substituted with anaryl group; “alkoxyalkyl” mean alkyl substituted with an alkoxy group;“cycloalkylalkyl” means alkyl substituted with cycloalkyl;“alkylcarbonyl” means —C(O)-A*, wherein A* is alkyl; “alkoxycarbonyl”means —C(O)—OA*, wherein A* is alkyl; and where alkyl is as definedabove. Alkoxy is preferably O(C₁-C₆) alkyl and includes methoxy, ethoxy,propoxy, butoxy, pentoxy and hexoxy.

“Cycloalkoxy” means a cycloalkyl-O— group wherein the cycloalkyl is asdefined above. Exemplary (C₃-C₇) cycloalkyloxy groups includecyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy and cycloheptoxy.

“Halogen” and “halo” are interchangeably used herein and each refers tofluorine, chlorine, bromine, or iodine.

The terms “haloalkyl”, “halocycloalkyl” and “haloalkoxy” mean alkyl,cycloalkyl, or alkoxy, as the case may be, substituted with one or morehalogen atoms. The term “halogen” or “halo” means F, Cl, Br or I.

“Acyl” refers to R″—C(O)—, where R″ is H, alkyl, substituted alkyl,heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl,aryl, alkylaryl, or substituted alkylaryl, and is indicated in thegeneral formula of a particular embodiment as “Ac”.

An “alkylene group” is represented by —[CH₂]_(z)—, wherein z is apositive integer, preferably from one to eight, more preferably from oneto four.

An “alkenylene group” is an alkylene in which at least a pair ofadjacent methylenes are replaced with —CH—CH—.

The term “(C₆-C₁₀) aryl” used alone or as part of a larger moiety as in“arylalkyl”, “arylalkoxy”, “aryloxy”, or “aryloxyalkyl”, meanscarbocyclic aromatic rings. The term “carbocyclic aromatic group” may beused interchangeably with the terms “aryl”, “aryl ring” “carbocyclicaromatic ring”, “aryl group” and “carbocyclic aromatic group”. An arylgroup typically has 6-10 ring atoms. A “substituted aryl group” issubstituted at any one or more substitutable ring atom. The term “C₆-C₁₆aryl” as used herein means a monocyclic, bicyclic or tricycliccarbocyclic ring system containing from 6 to 16 carbon atoms andincludes phenyl (Ph), naphthyl, anthracenyl,1,2-dihydronaphthyl1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like.The (C₆-C₁₀) aryl (C₁-C₆) alkyl group connects to the rest of themolecule through the (C₁-C₆) alkyl portion of the (C₆-C₁₀) aryl (C₁-C₆)alkyl group.

The term “Alkenyl” as used alone or as part of a larger moiety as in“Alkenylacyl” or “haloalkylacyl” means a straight or branchedhydrocarbon radical having a specified number of carbon atoms andincludes at least one double bond. An alkenyl group generally hasbetween 2 and 6 carbon atoms. The (C₆-C₁₀) aryl (C₂-C₆) alkenyl groupconnects to the remainder of the molecule through the (C₂-C₆) alkenylportion of (C₆-C₁₀) aryl (C₂-C₆) alkenyl.

“Alkenylacyl” refers to an acyl group, R″—C(O)—, where R″ is an alkenylor a substituted alkenyl (e.g., CH₃—CH═CH—C(O)—).

“Pharmaceutically acceptable carrier” means non-therapeutic componentsthat are of sufficient purity and quality for use in the formulation ofa composition of the invention that, when appropriately administered totypically do not produce an adverse reaction, and that are used as avehicle for a drug substance (e.g., a compound of Formula (I)).

“Intraperitoneal injection,” as used herein, refers to the injection ofa substance into the peritoneum (body cavity).

“Three times a day dosing”, as used herein, refers to threeadministrations of a pharmaceutical composition per every 24 hourperiod.

“Four times a day dosing” (QDS), as used herein, refers to fouradministrations of a pharmaceutical composition per every 24 hourperiod.

As used herein, “BDI” refers to twice daily. Further, as used herein,“QD” refers to once daily.

As used herein, “Cmin” refers to the minimum concentration that a drugachieves after the drug has been administered and prior to theadministration of a second or additional dose. Further, “Cmax”, as usedherein, refers to the maximum concentration. Similarly, “Tmax”, as usedherein, refers to the time of maximum concentration. Additionally,“AUC”, used herein, is the area under the concentration-time curve.Additionally, “50% effective concentration” (EC50), as used herein,refers to the concentration of an anti-viral that produces 50% of themaximal possible antiviral effect.

As used herein, the term “about” refers to a number that differs fromthe given number by less than 10%. In other embodiments, the term“about” indicates that the number differs from the given number by lessthan 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

The abbreviation “DENV”, as used herein, refers to dengue virus. Furtherthe four serotypes of the dengue virus are used herein as “DENV1”,“DENV2”, “DENV3” and “DENV4”.

“Antibody enhanced” or “Antibody Dependent Enhancement” (ADE), as usedherein interchangeably, refers to a DENV infection made more severe dueto a prior infection with one of the four DENV serotypes: DENV1, DENV2,DENV3, and DENV4.

As used herein, “dengue hemorrhagic fever” (DHF) and “dengue shocksyndrome” (DSS), refers to the severe and potentially fatal denguediseases that often occur during secondary heterotypic infections.

As used herein, “viral load” refers to the amount of virus in the bloodstream of a human subject.

Dosing Regimen

The present invention pertains to methods of treating a dengue virus(DENV) infection in a human subject, comprising administering to thehuman subject an initial dose of about 100 to about 600 mg of a compoundof Formula (I), or a pharmaceutical composition comprising a compound ofFormula (I), within from about onset of fever to about 72 hours of feveronset due to dengue infection and administering to the human subject adose of about 25 to about 300 mg of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), atintervals of from about 6 to about 12 hours until there is animprovement in the infection or between from about 1 day to about 10days.

In one embodiment, the compound of the invention is a compound ofFormula (I):

or a pharmaceutically acceptable salt there of;

wherein R¹, R², and R³ are independently H, (C1-C14) acyl, (C1-C14)alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14) haloalkylacyl (C1-C8)alkoxyacyl, or (C6-C10) arylacyl.

In another embodiment, R¹ and R² are H and R³ is a (C1-C14) acyl. Inanother embodiment, R¹ is CH₃—CH₂CH₂—C(O)—. In yet another embodiment,R² is CH₃—CH₂CH₂—C(O)—. In a further embodiment, R³ is CH₃—CH₂CH₂—C(O)—.In another embodiment, at least one but not more than two R1, R2, and R3is a hydrogen.

In yet another embodiment, the compound of the invention is a compoundof Formula (II):

or a pharmaceutically acceptable salt thereof.

The compound of Formula (II), or pharmaceutical composition comprising acompound of Formula (II), can be used in any of the embodiments providedherein for Formula (I).

In further embodiment, the compound of the invention is a compound ofFormula (III):

or a pharmaceutically acceptable salt there of;

wherein R¹, R², and R³ are independently H, (C1-C14) acyl, (C1-C14)alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14) haloalkylacyl (C1-C8)alkoxyacyl, or (C6-C10) arylacyl.

The compound of Formula (III), or pharmaceutical composition comprisinga compound of Formula (III), can be used in any of the embodimentsprovided herein for Formula (I).

The compounds of the invention useful for practicing the methodsdescribed herein may possess one or more chiral centers and so exist ina number of stereoisomeric forms. All stereoisomers and mixtures thereofare included in the scope of the present invention. Racemic compoundsmay either be separated using preparative HPLC and a column with achiral stationary phase or resolved to yield individual enantiomersmethods known to those skilled in the art. In addition, chiralintermediate compounds may be resolved and used to prepare chiralcompounds of the invention.

The compounds described herein may exist in one or more tautomericforms. All tautomers and mixtures thereof are included in the scope ofthe present invention.

The compounds of the present invention can be administered as the freebase or as a pharmaceutically acceptable salt. For example, an acid saltof a compound of the present invention containing an amine or otherbasic group can be obtained by reacting the compound with a suitableorganic or inorganic acid, resulting in pharmaceutically acceptableanionic salt forms. Examples of anionic salts include the acetate,benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calciumedetate, camsylate, carbonate, chloride, citrate, dihydrochloride,edetate, edisylate, estotate, esylate, furnaratc glyceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,lactobionate, malate, maleate, mandelate, mesylate, methylsulfate,mucate, napsylate, nitrate, pamoate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, teoclatc, tosylate,and triethiodide salts. In one embodiment, the compound of Formula (I)is a hydrochloride salt. In another embodiment, the compound of Formula(II) is a hydrochloride salt.

The invention is also directed to methods of the invention using apharmaceutical composition comprising a compound of Formula (I) orFormula (II). The disclosed compounds of Formula (I) and Formula (II)can be administered to the subject in conjunction with an acceptablepharmaceutical carrier or diluent as part of a pharmaceuticalcomposition for treatment of a dengue (DENV) infection, and according toany of the dosing regimens described herein. Formulation of the compoundto be administered will vary according to the route of administrationselected (e.g., solution, emulsion, capsule). Suitable pharmaceuticalcarriers may contain inert ingredients which do not interact with thecompound. Standard pharmaceutical formulation techniques can beemployed, such as those described in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceuticalcarriers for parenteral administration include, for example, sterilewater, physiological saline, bacteriostatic saline (saline containingabout 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank'ssolution, Ringer's-lactate and the like. Methods for encapsulatingcompositions (such as in a coating of hard gelatin or cyclodextran) areknown in the art (Baker, et al., “Controlled Release of Biological.Active Agents”, John Wiley and Sons, 1986).

In one embodiment, the pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a compound Formula(I). In another embodiment, the pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a compound Formula(II).

In preferred embodiments, the compound of Formula (I) is prodrug ofcastanospermine, a natural product derived from the seeds ofCastanospermum australe. Once administered compounds of Formula (I) arerapidly converted to castanospermine. Compounds of Formula (I) (e.g.,celgosivir) are more rapidly and efficiently absorbed thancastanospermine. Compounds of Formula (I) are also more readily absorbedinto cells. As a result, compounds of Formula (I) may have higher EC50values and in vivo efficacy than castanospermine against the dengue(DENV) virus.

Castanospermine has been shown to exert antiviral activity by inhibitinghost alpha-glucosidases I and II, enzymes essential for proper foldingof dengue-virus encoded glycoproteins such as E and prM. (6,7)Castanospermine targets dengue NS1 protein folding in dengue virusinfected cells. Impaired glycosylation of the NS1 protein leads toaccumulation of proteins in the endoplasmic reticulum (ER) anddrastically inhibits the virus replication. Since the drug target is ahost enzyme required for viral maturation, the potential for developmentof resistance is expected to be lower than a drug directed against aviral enzyme.

The methods of the invention treat a human subject having a dengue viralinfection. As used herein “treating” or “treatment” refers to obtainingdesired pharmacological and/or physiological effects. The effect caninclude achieving, partially or substantially one or more of thefollowing results: partially or totally reducing the extent of thedisease, disorder or syndrome; ameliorating or improving a clinicalsymptom or indicator associated with the disease, disorder or syndrome;delaying, inhibiting or decreasing the likelihood of the progression ofthe disease, disorder or syndrome.

The initial dose of a compound of Formula (I), or a pharmaceuticalcomposition comprising a compound of Formula (I), can be administered atany time within from about the onset of fever to about 72 hours afterfever onset due to dengue infection. In one embodiment, the initial doseis administered at the time of fever onset. In another embodiment, theinitial dose is administered within from about 24 hours of fever onset.In yet another embodiment, the initial dose is administered within fromabout 48 hours of fever onset. In a further embodiment, the initial doseis administered within from about 72 hours of fever onset. Subsequentdoses can be the same amount or vary to achieve steady state Cmin orplasma concentrations in the subject.

The human subject may be an adult or a child. As used herein, a “child”refers to a human subject who is between the ages of 1 day to 17 yearsof age. The term “adult” refers to a human subject who is 18 years ofage or older. Further, the plurality of human subjects may includeadults or children. In some embodiments, the plurality of human subjectsmay include only adults. In another embodiment, the plurality of humansubjects may include only children. In yet another embodiment, theplurality of human subjects may include both adults and children.

In another aspect, the present invention relates to a method of treatinga dengue invention in an adult subject comprising administering to theadult subject an initial dose of about 100 to about 600 mg of a compoundof Formula (I), or a pharmaceutical composition comprising a compound ofFormula (I), within from about onset of fever to about 72 hours of feveronset due to dengue infection and administering to the adult subject adose of about 25 to about 300 mg of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), atintervals of from about 6 to about 12 hours until there is animprovement in the infection or between about 1 day to about 10 days.

Example embodiments of initial and subsequent doses in an adult areshown in Table 1:

TABLE 1 Dosing Regimen for an Adult Initial Subsequent dose doseEmbodiment (mg)¹ (mg)^(2,3) 1 100 100 2 150 100 3 200 100 4 250 100 5300 100 6 350 100 7 400 100 8 450 100 9 500 100 10 550 100 11 600 100 12100 150 13 150 150 14 200 150 15 250 150 16 300 150 17 350 150 18 400150 19 450 150 20 500 150 21 550 150 22 600 150 23 600 175 24 100 200 25150 200 26 200 200 27 250 200 28 300 200 29 350 200 30 400 200 31 450200 32 500 200 33 550 200 34 600 200 35 100 250 36 150 250 37 200 250 38250 250 39 300 250 40 350 250 41 400 250 42 450 250 43 500 250 44 500250 45 550 250 46 600 250 47 100 300 48 150 300 49 200 300 50 250 300 51300 300 52 350 300 53 400 300 54 450 300 55 450 300 56 500 300 57 500300 58 550 300 59 600 300 ¹Initial dosing within about onset of fever toabout 72 hours. ²Subsequent dosing periodically from about 6 to about 12hours. ³Length of subsequent dosing, a) until infection improves, b)between about 1 day to 2 days, c) between about 2 to 3 days, d) betweenabout 3 to 4 days, d) between about 4 to 5 days, e) between about 5 to 7day, or f) between about 7 to 10 days.

In another aspect, the present invention relates to methods of treatinga dengue invention in an child subject comprising administering to thechild subject an initial dose of about 100 to about 600 mg of a compoundof Formula (I), or a pharmaceutics composition comprising a compound ofFormula (I), within from about onset of fever to about 72 hours of feveronset due to dengue infection and administering to the child subject adose of about 25 to about 300 mg of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), atintervals of from about 6 to about 12 hours until there is animprovement in the infection or between from about 1 day to about 10days.

Example embodiments of initial and subsequent doses combinations in achild are shown in Table 2:

TABLE 2 Dosing Regimen for a Child Initial Subsequent dose doseEmbodiment (mg)⁴ (mg)^(5,6) 1 25 25 2 50 25 3 75 25 4 100 25 5 150 25 6200 25 7 250 25 8 300 25 9 25 50 10 50 50 11 75 50 12 100 50 13 150 5014 200 50 15 250 50 16 300 50 17 25 75 18 50 75 19 75 75 20 100 75 21150 75 22 200 75 23 250 75 24 300 75 25 25 100 26 50 100 27 75 100 28100 100 29 150 100 30 200 100 31 250 100 32 300 100 33 25 150 34 50 15035 75 150 36 100 150 37 150 150 38 200 150 39 250 150 40 300 150 41 25200 42 50 200 43 75 200 44 100 200 45 150 200 46 200 200 47 250 200 48300 200 ⁴Initial dosing within about onset of fever to about 72 hours.⁵Subsequent dosing periodically from about 6 to about 12 hours. ⁶Lengthof subsequent dosing, a) until infection improves, b) between about 1day to 2 days, c) between about 2 to 3 days, d) between about 3 to 4days, d) between about 4 to 5 days, e) between about 5 to 7 day, or f)between about 7 to 10 days.

The human subject can be administered a compound of the presentinvention for a period of about between about 1 day to about 10 days. Inone embodiment, the subsequent dose of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), isadministered for about 1 day to about 2 days, in further embodiment, thesubsequent dose of a compound of Formula (I), or a pharmaceuticalcomposition comprising a compound of Formula (I), is administered forabout 2 to about 3 days. In another embodiment, the subsequent dose of acompound of Formula (I), or a pharmaceutical composition comprising acompound of Formula (I), is administered for about 3 to about 5 days. Inyet another embodiment, the subsequent dose of a compound of Formula(I), or a pharmaceutical composition comprising a compound of Formula(I), is administered for about 5 to about 7 days. In another embodiment,the subsequent dose of a compound of Formula (I), or a pharmaceuticalcomposition comprising a compound of Formula (I), is administered forabout 7 to about 10 days.

The present invention pertains to methods of treating a dengue virus(DENV) infection in a human subject, comprising administering to thehuman subject an initial dose of about 100 to about 600 mg of a compoundof Formula (I), or a pharmaceutical composition comprising a compound ofFormula (I), within from about onset of fever to about 72 hours of feveronset due to dengue infection; and administering to the human subject adose of about 25 to about 300 mg of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), atintervals of from about 6 to about 12 hours until there is animprovement in the infection or between from about 1 day to about 10days, wherein R¹, R², and R³ are independently H, (C1-C14) acyl,(C1-C14) alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14) haloalkylacyl(C1-C8) alkoxyacyl, or (C6-C10) arylacyl.

The invention is also directed to methods of treating a secondary dengueinfection in a human subject, comprising administering to the humansubject an initial dose of about 100 to about 600 mg of a compound ofFormula (I), or a pharmaceutical composition comprising a compound ofFormula (I), within from about onset of fever to about 72 hours of feveronset due to dengue infection; and administering to the human subject adose of about 25 to about 300 mg of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), atintervals of from about 6 to about 12 hours until there is animprovement in the infection or between from about 1 day to about 10days, wherein R¹, R², and R³ are independently H, (C1-C14) acyl,(C1-C14) alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14) haloalkylacyl(C1-C8) alkoxyacyl, or (C6-C10) arylacyl.

In one embodiment of the invention, the compound of Formula (I), is acompound of Formula (II) or a pharmaceutically acceptable salt thereof.

The invention also relates to methods of treating a dengue virus (DENV)infection in a human subject, comprising administering to the humansubject an initial dose of about 100 to about 600 mg of a compound ofFormula (II), or a pharmaceutical composition comprising a compound ofFormula (II), within from about onset of fever to about 72 hours offever onset due to dengue infection; and administering to the humansubject a dose of about 25 to about 300 mg of a compound of Formula(II), or a pharmaceutical composition comprising a compound or Formula(II), at intervals of from about 6 to about 12 hours until there is animprovement in the infection or between from about 1 day to about 10days.

In another aspect, the invention relates to methods of treating a denguevirus (DENV) infection in a human subject, comprising administeringorally to the human subject an initial dose of about 100 to about 600 mgof a compound of Formula (I), or a pharmaceutical composition comprisinga compound of Formula (I), within from about onset of fever to about 72hours of fever onset due to dengue infection; and administering orallyto the human subject a dose of about 25 to about 300 mg of a compound ofFormula (I), or a pharmaceutical composition comprising a compound ofFormula (I), at intervals of from about 6 to about 12 hours until thereis an improvement in the infection or between from about 1 day to about10 days, wherein R¹, R², and R³ are independently H, (C1-C14) acyl,(C1-C14) alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14) haloalkylacyl(C1-C8) alkoxyacyl, or (C6-C10) arylacyl.

In yet another aspect, the invention pertains to methods of treating adengue virus (DENV) infection in a human subject, comprisingadministering to the human subject an initial dose of about 100 to about600 mg of a compound of Formula (III), or a pharmaceutical compositioncomprising a compound of Formula (III), within from about onset of feverto about 72 hours of fever onset due to dengue infection; andadministering to the human subject a dose of about 25 to about 300 mg ofa compound of Formula (III), or a pharmaceutical composition comprisinga compound or Formula (III), at intervals of from about 6 to about 12hours until there is an improvement in the infection or between fromabout 1 day to about 10 days, wherein R¹, R², and R³ are independentlyH, (C1-C14) acyl, (C1-C14) alkenylacyl, (C3-C8) cycloalkylacyl, (C1-C14)haloalkylacyl (C1-C8) alkoxyacyl, or (C6-C10) arylacyl.

In certain embodiments of the invention, the human subject is an adultor a child. In further embodiments of the invention, the plurality ofhuman subjects may include adults or children. In some embodiments, theplurality of human subjects may include only adults, in anotherembodiment, the plurality of human subjects may include only children.In yet another embodiment, the plurality of human subjects may includeboth adults and children.

In certain embodiments of the invention, each of the plurality of humansubjects may be given a different dose. In another embodiment, each ofthe plurality of human subjects may be given the same dose. In furtherembodiment, each of the plurality of human subjects may be given avariety of doses. In yet another embodiment, some of the plurality ofthe human subjects may be given the same dose and some of the pluralityof the human subjects may be given a different dose.

In another embodiment, compound of Formula (I), the compound of Formula(II), or the compound of Formula (III) is converted to castanospermineafter administration to a human subject.

In yet another embodiment, a steady state Cmin serum or plasmaconcentration of between about 0.05 and about 2.0 microgram/mL ofcastanospermine in an adult or child human subject is attained.

In one embodiment of the invention, dengue viral infection comprises atleast one dengue virus selected from DENV1, DENV2, DENV3 and DENV4. Inanother embodiment, the dengue viral infection is secondary dengueinfection. In yet another embodiment of the invention, the human subjectto be treated is positive for a dengue infection using a NS1 strip assayor quantitative PCR. In further embodiment of the invention, thevirological log reduction in treated human subjects is at least 50%greater than untreated or placebo-treated groups. In yet anotherembodiment, administering the compound, or the pharmaceuticalcomposition, achieves a steady state Cmin serum or plasma concentrationof between about 0.05 and about 2.0 microgram/mL of castanospermine. Inanother embodiment of the invention, the compound, or the pharmaceuticalcomposition, is administered intravenously, orally, rectally orsublingually.

In one embodiment of the invention, the human subject is administered aninitial dose of 150 mg and a close of 100 mg is administered to thehuman subject every 6 hours for about 1 day. In another embodiment, thehuman subject is administered an initial dose, of 150 mg and a dose of100 mg is administered to the human subject every 6 hours for about 2days. In yet another embodiment, the human subject is administered aninitial dose of 150 mg and a close of 100 mg is administered to thehuman subject every 6 hours for about 5 days. In further embodiment, thehuman subject is administered an initial dose of 150 mg and a dose of100 mg is administered to the human subject every 8 hours for about 1day. In certain embodiment, the human subject is administered an initialdose of 150 mg and a dose of 100 mg is administered to the human subjectevery 8 hours for about 2 days. In another embodiment, the human subjectis administered an initial dose of 150 mg and a dose of 100 mg isadministered to the human subject every 8 hours for about 5 days. In yetanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 100 mg is administered to the human subject every12 hours for about 1 day. In another embodiment, the human subject isadministered an initial close of 150 mg and a dose of 100 mg isadministered to the human subject every 12 hours for about 2 days. Infurther embodiment, the human subject is administered an initial dose of150 mg and a dose of 100 mg is administered to the human subject every12 hours for about 5 days. In yet another embodiment, the human subjectis administered an initial dose of 150 mg and a dose of 150 mg isadministered to the human subject every 6 hours for about 1 day. Inanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 150 mg is administered to the human subject every 6hours for about 2 days. In yet another embodiment, the human subject isadministered an initial dose of 150 mg and a dose of 150 mg isadministered to the human subject every 6 hours for about. 5 days. Inanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 150 mg is administered to the human subject every 8hours for about 1 day. In certain embodiment, the human subject isadministered an initial dose of 150 mg and a close of 150 mg isadministered to the human subject every 8 hours for about 2 days. In yetanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 150 rag is administered to the human subject every8 hours for about 5 days. In one embodiment, the human subject isadministered an initial dose of 150 mg and a dose of 150 mg isadministered to the human subject every 12 hours for about 1 day. Infurther embodiment, the human subject is administered an initial dose of150 mg and a dose of 150 mg is administered to the human subject every12 hours for about 2 days. In yet another embodiment, the human subjectis administered an initial close of 150 mg and a dose of 150 mg isadministered to the human subject every 12 hours for about 5 days. Inanother embodiment, the human subject is administered an initial dose of150 mg and a close of 200 mg is administered to the human subject every6 hours for about 1 day. In one embodiment, the human subject isadministered an initial dose of 150 mg and a dose of 200 mg isadministered to the human subject every 6 hours for about 2 days. Inanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 200 mg is administered to the human subject every 6hours for about 5 days. In further embodiment, wherein the human subjectis administered an initial dose of 150 mg and a dose of 200 mg isadministered to the human subject every 8 hours for about 1 day. In yetanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 200 mg is administered to the human subject every 8hours for about 2 days. In one embodiment, the human subject isadministered an initial dose of 150 mg and a dose of 200 mg isadministered to the human subject every 8 hours for about 5 days. Inanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 200 mg is administered to the human subject every12 hours for about 1 day. In further embodiment, the human subject isadministered an initial dose of 150 mg and a dose of 200 mg isadministered to the human subject every 12 hours for about 2 days. Inanother embodiment, the human subject is administered an initial dose of150 mg and a dose of 200 rug is administered to the human subject every12 hours for about 5 days. In certain embodiment, the human subject isadministered an initial dose of 200 mg and a dose of 100 mg isadministered to the human subject every 6 hours for about 1 day. In yetanother embodiment, the human subject is administered an initial dose of200 mg and a dose of 100 mg is administered to the human subject every 6hours for about 2 days. In another embodiment, the human subject isadministered an initial dose of 200 mg and a dose of 100 mg isadministered to the human subject every 6 hours for about 5 days. In oneembodiment, the human subject is administered an initial dose of 200 mgand a dose of 100 mg is administered to the human subject every 8 hoursfor about 1 day. In further embodiment, the human subject isadministered an initial dose of 200 mg and a dose of 100 mg isadministered to the human subject every 8 hours for about 2 days. In yetanother embodiment, the human subject is administered an initial dose of200 mg and a dose of 100 mg is administered to the human subject every 8hours for about 5 days. In another embodiment, the human subject isadministered an initial dose of 200 mg and a dose of 100 mg isadministered to the human subject every 12 hours for about 1 day. In oneembodiment, the human subject is administered an initial dose of 200 mgand a dose of 100 mg is administered to the human subject every 12 hoursfor about 2 days. In another embodiment, the human subject isadministered an initial dose of 200 mg and a dose of 100 mg isadministered to the human subject every 12 hours for about 5 days. Infurther embodiment, the human subject is administered an initial dose of200 mg and a dose of 150 mg is administered to the human subject every 6hours for about 1 day. In another embodiment of the invention, the humansubject is administered an initial dose of 200 mg and a dose of 150 mgis administered to the human subject every 6 hours for about 2 days. Inyet another embodiment, the human subject is administered an initialdose of 200 mg and a dose of 150 mg is administered to the human subjectevery 6 hours for about 5 days. In another embodiment, the human subjectis administered an initial dose of 200 mg and a dose of 150 mg isadministered to the human subject every 8 hours for about 1 day. In oneembodiment, the human subject is administered an initial dose of 200 mgand a dose of 150 mg is administered to the human subject every 8 hoursfor about 2 days. In yet another embodiment, the human subject isadministered an initial dose of 200 mg and a dose of 150 mg isadministered to the human subject every 8 hours for about 5 days. Inanother embodiment, the human subject is administered an initial dose of200 mg and a dose of 150 mg is administered to the human subject every12 hours for about 1 day. In another embodiment, the human subject isadministered an initial dose of 200 mg and a dose of 150 mg isadministered to the human subject every 12 hours for about 2 days. In anembodiment of the invention, the human subject is administered aninitial dose of 200 mg and a dose of 150 mg is administered to the humansubject every 12 hours for about 5 days. In further embodiment, thehuman subject is administered an initial dose of 200 mug and a dose of200 mg is administered to the human subject every 6 hours for about 1day. In yet another embodiment, the human subject is administered aninitial dose of 200 mg and a dose of 200 mg is administered to the humansubject every 6 hours for about 2 days. In another embodiment, the humansubject is administered an initial dose of 200 mg and a dose of 200 mgis administered to the human subject every 6 hours for about 5 days. Inan embodiment of the invention, the human subject is administered aninitial dose of 200 mg and a dose of 200 mg is administered to the humansubject every 8 hours for about 1 day. In further embodiment, the humansubject is administered an initial close of 200 ma and a dose of 200 mgis administered to the human subject every 8 hours for about 2 days. Inyet another embodiment, the human subject is administered an initialdose of 200 mg and a dose of 200 mg is administered to the human subjectevery 8 hours for about 5 days. In another embodiment, the human subjectis administered an initial dose of 200 mg and a dose of 200 mg isadministered to the human subject every 12 hours for about 1 day. In anembodiment of the invention, the human subject is administered aninitial dose of 200 mug and a dose of 200 mg is administered to thehuman subject every 12 hours for about 2 days. In one embodiment, thehuman subject is administered an initial dose of 200 mg and a dose of200 mg is administered to the human subject every 12 hours for about 5days. In another embodiment, the human subject is administered a singleor a divided dose of about 25 to about 300 mg of the compound or thepharmaceutical composition, for about between about 5 days to about 10days. In yet another embodiment, the human subject is administered adivided dose of about 25 to about 300 mg of the compound, or thepharmaceutical composition, for about between about 5 days to about 10days.

The compounds of the present invention can be administered in a singleor a divided dose. In one embodiment, the human subject is administereda divided dose of about 25 to about 300 mg of a compound of Formula (I),or a pharmaceutical composition comprising a compound of Formula (I),for about between about 1 day to about 10 days. In another embodiment,the human subject is administered a single dose of about 25 to about 300mg of a compound of Formula (I), or a pharmaceutical compositioncomprising a compound of Formula (I), for about between about 1 day toabout 10 days.

Compounds of Formula (I) and Formula (II) inhibit the replication of avariety of laboratory and clinical dengue strains of DEN V1-4, withsubmicromolar EC50 values. As used herein, “EC50” refers to theconcentration of an anti-viral that produces 50% of the maximal possibleantiviral effect. In one embodiment, the method of the invention is usedto treat an infection caused by at least one dengue virus selected fromDENV1, DENV2, DENV3, and DENV4.

The method of the present invention is used to treat a dengue viralinfection in a subject who has tested positive for a dengue virus. Knownmethods for diagnosis of dengue viral infection can be used including,but not limited to, an NS1 strip assay or a quantitative PCR assay. Theselected method should be rapid enough for a diagnosis within from aboutonset of fever to about 72 hours of fever onset to optimize thetherapeutic regimen of the various embodiments of the invention.

In one embodiment, the human subject tests positive for a dengueinfection in a NS1 strip assay. In another embodiment, the human subjecttests positive for a dengue infection in a quantitative PCR assay.

The compounds of the present invention can be administeredintravenously, orally, rectally or sublingually. Intravenous, oral,rectal and sublingual dosing can be in a single or divided dose.Intravenous dosing can also be a slow infusion over a period of time andthe slow infusions can be constant or intermittent.

The compounds of the invention can be administered several times a dayor as needed to maintain a steady Cmin serum concentration of betweenabout 0.05 and about 2.0 microgram/mL. A suitable interval between thetwo administrations includes any time period which maintains atherapeutically effective plasma level of a compound of Formula (I).Such an interval can be, for example, about 12 hours. In one embodiment,the human subject is administered a compound of Formula (I) or acompound of Formula (II) twice a day. Dosing at intervals is intended tocover subsequent dosing, either as a single or a divided dose, routinelyduring the course of therapy. For example, dosing can be about every 6to about 12 hours during the course of treatment, but it is intended tocover the possibility that a dose may have been missed during at leastone interval. In another embodiment, the human subject is administered acompound of Formula (I) or a compound of Formula (II) three times a day.In yet another embodiment, the human subject is administered a compoundof Formula (I) or a compound of Formula (II) four times a day.

As such, a first dose can be administered at 6 am on Day 1 and a seconddose can be administered at 6 pm on Day 1 for a total of two doses in a24 hour period or day. Further, a first dose can be administered at 12am on Day 1, a second dose can be administered at 6 am on Day 1, a thirddose can be administered at 12 pm on Day 1, and a fourth dose can beadministered at 6 pm on Day 1 for a total of four doses in a 24 hourperiod or day. In one embodiment, the therapeutically effective plasmalevel is the level at which the Cmin concentration of a compound ofFormula (I) is achieved.

The invention also relates to a method of treating a dengue virusinfection by achieving a steady state Cmin serum or plasma concentrationof between about 0.05 and about 2.0 microgram/mL of castanospermine inan adult or child human subject. As used herein, “Cmin” refers to theminimum concentration that a drug achieves after the drug has beenadministered and prior to the administration of a second or additionaldose. Steady state Cmin is achieved when the overall intake of a drugCmin concentration is fairly in dynamic equilibrium with itselimination. In some embodiments, Cmin concentration of castanospermineis determined at one or more points following treatment with techniquesknown in the art.

In one embodiment, the steady state Cmin serum or plasma concentrationachieved in an adult or child subject is between about 0.08 and about0.5 microgram/mL of castanospermine. In another embodiment, the steadystate Cmin serum or plasma concentration achieved. In an adult or childsubject is between about 0.05 and about 0.08 microgram/mL ofcastanospermine. In yet another embodiment, the steady state Cmin serumor plasma concentration achieved in an adult or child subject is betweenabout 0.08 and about 0.11 microgram/mL of castanospermine. In a furtherembodiment, the steady state Cmin serum or plasma concentration achievedin an adult or child subject is between about 0.11 and about 0.3microgram/mL of castanospermine. In another embodiment, the steady stateCmin serum or plasma concentration achieved in an adult or child subjectis between about 0.3 and about 0.75 microgram/mL of castanospermine. Infurther embodiment, the steady state Cmin serum or plasma concentrationachieved in an adult or child subject is between about 0.75 and about1.0 microgram/mL of castanospermine. In yet another embodiment, thesteady state Cmin serum or plasma concentration achieved in an adult orchild subject is between about 1.0 and about 2.0 microgram/mL ofcastanospermine. In another embodiment, the steady state Cmin serum orplasma concentration achieved in an adult or child subject is betweenabout 1.0 and about 1.5 microgram/mL of castanospermine. In furtherembodiment, the steady state Cmin serum or plasma concentration achievedin an adult or child subject is between about 1.5 and about 2.0microgram/mL of castanospermine. In yet another embodiment, the steadystate Cmin serum or plasma concentration achieved in an adult or childsubject is between about 1.25 and about 1.75 microgram/mL ofcastanospermine.

The invention further relates to a method of treating a dengue virusinfection by achieving an average steady state Cmin serum or plasmaconcentration of between about 0.05 and about 2.0 microgram/mL ofcastanosperine in a plurality of human subjects after the administrationof the compounds of the invention. The average steady state Cmin of theplurality of human subjects is calculated as an average of steady stateCmin serum or plasma concentrations from each of the plurality of humansubjects. In certain embodiments of the invention, an average steadystate Cmin serum or plasma concentration of between about 0.05 and about2.0 microgram/mL of castanospermine in the plurality of human subjectsis attained after treatment.

In one embodiment, the average steady state Cmin serum or plasmaconcentration achieved in the plurality of human subjects is betweenabout 0.08 and about 0.5 microgram/mL of castanospermine. In anotherembodiment, the average steady state Cmin serum or plasma concentrationachieved in the plurality of human subjects is between about 0.05 andabout 0.08 microgram/mL of castanospermine. In yet another embodiment,the average steady state Cmin serum or plasma concentration achieved inthe plurality of human subjects is between about 0.08 and about 0.11microgram/mL of castanospermine. In a further embodiment, the averagesteady state Cmin serum or plasma concentration achieved in theplurality of human subjects is between about 0.11 and about 0.3microgram/mL of castanospermine. In another embodiment, the averagesteady state Cmin serum or plasma concentration achieved in theplurality of human subjects is between about 0.3 and about 0.75microgram/mL of castanospermine. In further embodiment, the averagesteady state Cmin serum or plasma concentration achieved in theplurality of human subjects is between about 0.75 and about 1.0microgram/mL of castanospermine. In yet another embodiment, the averagesteady state Cmin serum or plasma concentration achieved in theplurality of human subjects is between about 1.0 and about 2.0microgram/mL of castanospermine. In another embodiment, the averagesteady state Cmin serum or plasma concentration achieved in theplurality of human subjects is between about 1.0 and about 1.5microgram/mL of castanospermine, in further embodiment, the averagesteady state Cmin serum or plasma concentration achieved in theplurality of human subjects is between about 1.5 and about 2.0microgram/mL of castanospermine. In yet another embodiment, the averagesteady state Cmin serum or plasma concentration achieved in theplurality of human subjects is between about 1.25 and about 1.75microgram/mt of castanospermine.

In another aspect, methods of the present invention can treat asecondary dengue infection or an “antibody enhanced” (ADE) dengueinfection. A “secondary” infection refers to a DENV infection in apatient who was previously infected with DENV. An “antibody enhanced”infection refers to a DENV infection made more severe due to a priorinfection with one of the four DENV serotypes. Ninety percent (90%) ofsevere and potentially fatal dengue diseases, such as dengue hemorrhagicfever (DHF) or dengue shock syndrome (DSS) are caused by a secondarydengue infection. In secondary dengue infection, prior infection causesgeneration of an antibody that takes on a pathogenic role. Uponreinfection with DENV, the antibody response triggers a systemicinflammatory reaction resulting in vascular leakage. In one embodiment,the dengue viral infection is a secondary dengue infection.

In another aspect of the invention, viral load reduction of a treatedhuman subject is at least 50% greater than untreated or placebo-treatedhuman subjects. As used herein, the term “viral load” refers to theamount of virus in the blood stream of a human subject. The viral loadis measured before administration of the first close and then at varioustime intervals after administration. The dose amounts can be adjusted toincrease the viral load reduction or if no viral reduction is observedat a specific dose it can be adjusted to promote viral reduction.

In one embodiment, the virological log reduction in human subjectstreated with a compound of Formula (I) is at least about 50% greaterthan untreated or placebo-treated groups. In another embodiment, thevirological tog reduction in human subjects treated with a compound ofFormula (I) is between about 60 to 70% greater than untreated orplacebo-treated groups. In another embodiment, the virological logreduction in human subjects treated with a compound of Formula (I) isbetween about 70 to 80% greater than untreated or placebo-treatedgroups. In yet another embodiment, the virological log reduction inhuman subjects treated with a compound of Formula (I) is between about80 to 90% greater than untreated or placebo-treated groups.

EXAMPLES Example 1: Cell-Based Flaviviral Immunodetection Assay (CH)

BHK21 cells were seeded at 1.3×104 in 96-well plate and incubatedovernight at 37° C. in 5% CO₂ incubator. Confluent monolayer of BHK21cells were infected with DENV2 (TSV01 strain) at an MOI (multiplicity ofinfection) 0.3 in the presence of various concentrations of testcompounds and incubated for 1 hour at 37° C. in 5% CO₂ incubator,Infected cells were incubated with test compounds for another 48 hoursat 37° C. in 5% CO₂. Cells were fixed with methanol and mouse monoclonalantibody 4G2 was used to detect DENV E protein, which was quantifiedusing a secondary anti-mouse antibody conjugated with horseradishperoxidase (HRP). Absorbance was read at 450 nm and dose-response curvewas plotted accordingly. The 50% effective concentration (EC50), thatis, the concentration of the test compound that decreased the level ofviral E protein production by 50%, was calculated by nonlinearregression analysis.

Celgosivir inhibits virus production in a concentration-dependent mannerwith an EC50 0.22 μM for DENV2 (FIG. 1). The EC50 values for the otherthree serotypes are also in the sub-micromolar range (0.31 to 0.65 μM).Castanospermine (CAST) has an EC50 of ˜21 μM against DENV2, which may beattributed to its lower uptake into cells compared with celgosivir.

The results are shown in Table 3.

TABLE 3 EC50 values of celgosivir hydrochloride on DENV 1-4 infection inthe CFI assay. Dengue Serotype CFI (EC50) Compound DENV1 0.65 ± 0.16 μMCelgosivir DENV2 0.22 ± 0.01 μM Celgosivir DENV3 0.68 ± 0.02 μMCelgosivir DENV4 0.31 ± 0.12 μM Celgosivir DENV2 ~21 μM Castanospermine

Example 2: Fluorescence Microscopy

BHK21 cells were infected with DENV2 and treated with 20 celgosivir orsaline. After 24 hr incubation, cells were stained with nuclear staindiamidino-2-phenylindole (DAPI) or with a MAb against NS1 in conjunctionwith a secondary antibody conjugated with Alexa-488 (green), thenexamined by fluorescent microscopy for the presence of NS1, a marker forviral replication. Viral-infected cells show abundant NS1 in thecytoplasm, whereas those treated with celgosivir have almost completesuppression of viral replication (FIG. 2).

Other cellular microscopic studies, which used specific antibodies thatare markers for the endoplasmic reticulum (ER) or Golgi (BiP/Grp78) andGigantin, respectively, were conducted and demonstrated thatnonglycosylated NS1 accumulates in the ER and does not transport throughthe trans-Golgi network in order for it to be processed for secretion.

Celgosivir treatment also results in upregulation of pro-survival hostgene products such as EDEM and XBP1, while apoptotic markers such asCHOP are down-regulated during drug-induced unfolded protein response(data not shown). The pro-survival products enhance the clearance ofmisfolded proteins by directing them to the proteosome for degradation.

Example 3: Replicon Assay

The DENV subgenomic replicon was derived from the DENV2 strain NGC andconsists of a luciferase reporter and a puromycin resistance gene forstable transfection. (7) Only the nonstructural proteins NS1, NS2A,NS2B, NS3, NS4A, NS4B, and NS5 were represented in the repliconconstruct. Of the nonstructural proteins, only NS1 is known to beglycosylated.

The stably transfected A549 replicon cell line was plated into a 96-wellplate in the presence of various concentrations of test compounds andincubated for 48 hour at 37° C. in 5% CO₂ incubator. The luciferaseactivity of the replicon treated with various concentrations of the testcompound was measured using EnduRen (Promega) at a 1:1000 dilutionfollowed by incubation for 2 hrs before the luminescence was read usinga Tecan plate-reader. The data was plotted against the logtransformation of the concentration of the compounds to obtain adose-response curve to calculate the EC50 value. Celgosivir inhibitedreplicon production in a concentration-dependent manner, with an EC50value of 2.2 μM (FIG. 3). This data shows inhibition of hostalphaglucosidase activity by celgosivir targets both structural andnon-structural dengue proteins and severely affects viral assembly andreplication.

Example 4: ADE Infection of Human Monocytes

The activity of celgosivir in infected human monocytes, one of theprimary circulating cell types infected by dengue virus, was examined.THP-1 cells were grown in RPMI-1640 maintenance medium containing 10%fetal calf serum and 1% Penicillin-streptomycin and cultured in 37° C.incubator supplemented with 5% CO₂. For ADE infection in THP-1 cells,virus (DENV2, MOI-10) was mixed with sub-neutralizing concentration ofhumanized 4G2 monoclonal antibody (0.05 μg/0.5 ml) and allowed toincubate on ice for 1 hr in the serum free medium, to allow immunecomplex formations. The immune complex was then added on to THP-1(1×10⁵) cells per well in a 24 well plate and further incubated for 2hours at 37° C. incubator. An excess unbound immune complex was thenremoved by spinning cells at 1000 rpm for 5 min and then cells weresupplemented with complete growth maintenance medium. For drug testingin the ADE condition, cells were mock (untreated) or celgosivir-treatedfor 48 hours, and finally media supernatant was collected for plaqueassay analysis.

Addition of MAb against protein E results in a nearly 2 log increase inviremia (FIG. 4A). After addition of varying concentrations ofcelgosivir, viral replication was suppressed with an EC50 of 0.5 μM(FIG. 4B). Under the same conditions, Castanospermine was shown tosuppress viral replication with an EC50 of 14 μM.

Example 5: Animal Models Mouse Viremia Models

To develop an in vivo test system for anti-dengue drugs, immunocompetentSv/129 mice deficient in type I and II interferon receptors (AG129),were injected with an unadapted, clinical strain of DENV2, resulting ina dose-dependent transient viremia lasting several days and peaking onday 3 after infection. (8) NS1 protein, proinflammatory cytokines, andneutralizing IgM and IgG antibodies were detected and also the mice hadsplenomegaly. (8) Selected compounds such as celgosivir significantlyreduced viremia in a dose-dependent manner, even after delayedtreatment, leading to a reduction of splenomegaly and proinflammatorycytokine levels. Thereby validating dengue this mouse model as asuitable system for testing anti-dengue drugs. Furthermore, theyindicate that antiviral treatment during the acute phase of dengue fevercan reduce the severity of the disease. (8) In the AG129 mouse model,celgosivir significantly reduced viremia by 88% and 55%, respectively,when given at the time of infection or after treatment was delayed by 24hours.

To model ADE, mice were injected IP (intraperitoneal) with 15 mg ofmouse monoclonal antibody against the (DENV) E protein (4G2 clone) oneday prior to infection. (9) For treatment, celgosivir was injected IPonce or twice daily for a total of 5 days. Mice were monitored every daytill Day 12 post infection. The data were plotted as Kaplan-Meier curvesusing Prism 5.0 software.

In this lethal model of viremia, untreated mice had 0% survival by Day5, whereas mice treated with 50 mg/kg celgosivir PO (oral) BID (twice aday), showed 100% survival even at Day 12 (p=0.0001). Even whentreatment was delayed 24 and 48 hr, celgosivir was still able to conferprotection, with 75% and 50% survival, respectively at Day 12. Dosingstudies at 10, 25, and 50 mg/kg PO BID resulted in 12%, 62% and 100%survival. respectively. Surprisingly when mice were treated once dailyat 100 mg/kg PO, no mice survived past day 6 compared to 100% Survivalat 50 mg/kg twice daily, indicating that the drug, when divided into twodoses, is more effective than giving, the same total dose once a day.

Example 6: Dosing and Schedule Effects

To evaluate the effect of dose and dose schedule, four drug regimenswere studied in the ADE viremia model: 10, 25 and 50 mg/kg BID and 100mg/kg QD (once daily). For the BID schedule, survival increased withincreasing dose with survival rates of 13%, 63% and 100% for 10, 25, and50 mg/kg BID. Survival rates were also sensitive to schedule −50 mg/kgBID dosing achieved complete protection, but the same total dose of 100mg/kg given as a single daily dose failed to protect animals from death,with none surviving past Day 6. Even 25 mg/kg given BID was moreeffective than 100 mg/kg QD. Hence, dividing the dose was more effectivethan a larger daily dose.

Delayed Dosing

Even when treatment was delayed (FIG. 5A), celgosivir (50 mg/kg BID)exerted a protective effect. In this experiment, survival at Day 12 was100%, 75%, and 50% when treatment was immediate, delayed 24 hr ordelayed 48 hr after DENV2 infection, respectively, in the ADE model; bycontrast, none (0%) of the sham-treated mice survived past day 5 (FIG.5B). Viremia, as determined by plaque assay, in celgosivir-treated mice(50 mg/kg BID) was 8.3% of levels in sham-treated mice after only 1 dayof dosing. By study day 3, viremia had increased in untreated mice bymore than 7-fold. Celgosivir demonstrated significantly lower viremia intreated mice—levels were 17%, 26% and 69% of levels in sham-treated micewhen treatment was immediate, delayed 24 hr, or delayed 48 hr,respectively. The results also demonstrate that lower viremia isassociated with improved outcome.

Example 7: Pharmacokinetics Mouse Studies

Celgosivir is more rapidly and efficiently absorbed thancastanospermine. In the mouse, single-dose celgosivir (25 mg/kg PC)demonstrated a 5-fold greater uptake into the plasma at 2 and 5 minpost-dosing than a single dose of Castanospermine (16 mg/kg PO). At 5min after administration of celgosivir, only castanospermine wasdetected in the plasma, confirming the rapid conversion of celgosivirinto castanospermine.

The concentration profile of celgosivir and castanospermine in the mouseafter a single IP dose of celgosivir at a dose of 50 mg/kg isillustrated in FIG. 6A. Serum concentrations were measured using avalidated LC/MS/MS assay performed. Celgosivir was measurable in serumonly up to 1 hr post-dosing. Conversion to castanospermine is rapid, andthe maximal castanospermine concentration (Cmax) of 31.6 μg/mL occurs atthe first sampling time of 10 min post-dosing. AUC was 22.8 μg·hr/ml.Apparent clearance (CL/F) was 2.2 L/h/kg, mean residence time was 3.5hr, and the terminal half-life was 5.0 hr.

The castanospermine concentration profile after dosing at 50 mg/kg BIDfor 5 days was calculated by nonparametric superposition (FIG. 6B). Thisdosing regimen of celgosivir protected 100% of mice in the lethal ADEdengue infection model. Steady state Cmin and Cmax were 0.4 μg/mL and26.4 μg/mL, respectively.

Rat studies have been published. The published data shows that followinga single dose of celgosivir in healthy rats at 35 mg/kg PO, Cmax, Tmaxand AUC values were 8.76±1.15 μg/ml, 0.44±0.01 brand 10.5 μg·hr/ml,respectively. (10) Oral bioavailability in the rat was 93%, oralclearance (CL/F) was 2.5 L/h/kg, and half-life was 2.7 hr. Theanti-diarrhea agent loperamide had no effect on the PK in either normalrats or those with castor oil-induced diarrhea. (11)

In rats administered radiolabeled castanospermine, the majority ofradioactivity was detected within urine after PO (oral) or IV dosingwithin 24 hr, and more than 92% was identified as castanospermine. (12)

In rats with portal vein catheters administered 400 mg/kg celgosivir PO,celgosivir concentrations in the portal vein were approximately 4.4 to13.2-fold higher than those in peripheral circulation. The mean Cmax ofcelgosivir in the portal vein was 1.9 μg/ml compared with 105.6 μg/mLfor castanospermine. AUC 0-60 min was 1.0 μg·hr/ml for celgosivir vs,63.9 μg·hr/ml for castanospermine. (10) Hence the majority of celgosivirwas converted to castanospermine prior to liver exposure, probably inthe gastrointestinal tract.

The maximum tolerated dose) in humans was determined in studiesconducted as part of HIV drug trials. The MTD was 400 mg per day for 184days. (13)

Example 8: Effect of Diet on Gastrointestinal Effects of GlucosidaseInhibitors

Mice given a high dose of castanospermine (2 g/kg), when fed withsucrose- and starch containing diets develop severe diarrhea and highintestinal bacterial counts. (14) This is believed to be due tooff-target inhibition of gastrointestinal α-amylase, sucrase, maltaseand other enzymes involved in sucrose and glycogen breakdown. Celgosiviris a weaker inhibitor than castanospermine of the above-mentionedgastrointestinal enzymes. (10) When mice were fed a diet of proteins,vitamins and glucose and which did not contain sucrose or other complexsugars and carbohydrates, the GI toxicity of castanospermine wasameliorated.

Example 9: Safety Pharmacology Human Safety Pharmacology

Previous studies have provided valuable information on the clinicalpharmacology, pharmacokinetics, safety and tolerability of celgosivir inhumans. These studies have included single and multiple dosing as wellas QD dosing ranging from 10 to 600 mg for 2 to 12 weeks.

In humans, celgosivir has been shown to rapidly convert tocastanospermine where only the latter was detected in plasma. Thecelgosivir was also shown to rapidly absorb with peak concentrations ofcastanospermine occurring between 0.3 and 1.3 hr. Cmax and AUC were alsoshown to increase in proportion to dose. The terminal half-life forcelgosivir in humans was 18 hr, and oral clearance was 12 L/hr. (15)

Celgosivir is rapidly converted to castanospermine, which is excretedunchanged in the urine, with no other metabolites detected. Adverseevents were largely gastrointestinal, namely flatulence and diarrhea, ofmild to moderate intensity. Asymptomatic elevations in serum creatinekinase were also observed, which were reversed within 2 weeks afterdiscontinuation of the drug. No serious adverse events have beenreported.

Example 10: Treatment of a Dengue Infection in an Adult Subject withCelgosivir Hydrochloride

A human subject was orally administered an initial loading dose of 400mg of celgosivir followed by a maintenance dose of 200 mg every 12 hrfor a total of nine (9) doses. Drug was taken by the human subjectwithout food, i.e., 1 hr before consuming food or 2 hr afterwards.Subject was also placed on a special diet of protein, vitamins, andglucose containing minimal complex sugars or starches to minimize thelikelihood of gastrointestinal side effects. During treatment, severalserum and urine samples were obtained to analyze for drug levels.

On fifth day of treatment, after blood draws, administration of the lastdose, safety assessments, determination of risk for DHF or DSS, andsatisfactory clinical status, the human subject were discharged. Bloodsampling and safety assessments were then made on Days 7, 10, and 15.Human subjects completed visual analog scales for joint and muscle pain(VASP), and were evaluated for safety and tolerability daily duringtreatment and at each subsequent visit. Virological log reduction (VLR)was examined from Day 2 to 4. Viral clearance, serum NS1 levels,leukocyte and platelet count, and, hemoconcentration were alsodetermined.

Example 11:Pharmacokinetic Model

Population pharmacokinetic (PK) analysis of castanospermine plasmaconcentration data after administration of celgosivir was performedusing the non-linear mixed effects modeling software NONMEM (version7.2.0), (16) Pharmacokinetic parameters were estimated using the firstorder conditional estimation method (FOCE) with eta-epsilon interaction.Due to limited data during the input phase, first-orderabsorption/formation of castanospermine was assumed. Between-subjectvariability was assumed to be log-normally distributed. Modeldevelopment was guided by objective function value (OFV), precision ofthe parameter estimates, inspection of standard goodness of fit plotsand scientific plausibility. The empirical Bayes estimates from thefinal model were used to predict individual concentrations for astandard saturated sampling design (i.e., samples collected at 0, 0.25,0.5, 1, 1,5, 2, 4, 6 and 12 hours post-dose)) after the first dose (400mg) and dose 9 (day 5; 200 mg). These data were then use to determinethe non-compartmental PK analysis (NCA) parameters area under theconcentration-time curve over the dosing interval (AUC_(tau)), maximumconcentration (C_(max)), time of maximum concentration (T_(max)) andminimum concentration (C_(min)). The NCA parameter values were derivedusing the statistical analysis software R (version 3.0.1). (17) NCAparameters after dose 9 were only determined for subject who receivedthis dose. The mean NCA PK parameter values used to estimate themodel-dependent parameter values were taken from the results of a PhaseI trial investigating celgosivir and castanospermine PK after multipledoses (10-450 mg/day for 14 days) of celgosivir in HIV-positivepatients. (18, 19)

These data are reproduced in Table 4.

TABLE 4 NCA PK Parameter Values Dose AUC_(ss) C_(max, ss) t_(max)C_(min, ss) t_(1/2) (mg) (mg · h/L) (ng/mL) (h) (ng/mL) (h) 10 1.000.2266 0.7 8 13 20 1.75 0.52 1 11 27 40 3.61 1.07 1 23 30 80 6.53 1.720.3 29 16 160 13.30 3.73 0.8 70 21 240 21.50 5.76 0.6 110 16 360 32.106.319 0.7 179 15 450 39.80 10.50 NR 191 14 Note: The 80 and 450 mgdosing groups were excluded from the analysis conducted. AUC_(ss) isarea under the concentration-time curve at steady-state, C_(max, ss) isthe maximum concentration at steady-state, t_(max) is the time ofmaximum concentration, C_(min) is the minimum concentration atsteady-state, t_(1/2) is the terminal elimination half-life. NRrepresents values that were not reported.

A sparse PK sampling design was employed with plasma samples collectedat pre-dose and 23, 25, 47, 49.5, 71, 74 and 95 hours after first doseadministration. (20) A total of 50 otherwise healthy patients who haddengue fever were enrolled and randomly assigned in a 1:1 ratio toreceive either celgosivir or placebo. Active treatment participantsreceived a 400 mg loading dose followed by 200 mg every 12 hours for 9doses (i.e. 5 days of treatment). A total of 163 concentrations from 24individuals were available for analysis, Pharmacokinetic data were bestdescribed by a 1-compartment model with first-order input andelimination. No time-dependent changes in PK were observed. A plot ofmeasured concentrations overlayed with the 10^(th) to 90^(th) predictioninterval from data simulated for 2000 individuals demonstrates the modeladequately describes the data and is illustrated in FIG. 7. Thepredicted individual NCA parameter values after doses 1 and 9 aresummarised in Table 5. No major accumulation was observed, with meanexposure (i.e. C_(max) and AUC_(tau)) on day 5 (dose 9; 200 mg) lessthan that after first dose (400 mg). Variability in exposure betweensubjects was considered small to moderate.

TABLE 5 Predicted non-compartmental pharmacokinetic parameters afterdoses 1 and 9. Dose 1 Dose 9 (400 mg; 0-12 h) (200 mg; 96-108 h)Parameter [n = 24] [n = 22] AUC_(tau) Mean (SD) 48691 (2980) 25703(1912) (ng · h/mL) Median (Range) 48125 (42107, 54821) 25384 (21727,28977) Cmax Mean (SD) 9703 (129) 5127 (135) (ng/mL) Median (Range) 9684(9394, 9950) 5105 (4844, 5422) Tmax Mean (SD) 1 (na) 1 (na) (h) Median(Range) 1 (1, 1) 1 (1, 1) Cmin Mean (SD) 684 (153) 357 (85) (ng/mL)Median (Range) 647 (385, 1025) 339 (198, 555)

Plots of simulated population mean castanospermine concentration-timeprofiles after various celgosivir dosing regimens are presented in FIG.8A-H. The regimen that best produced trough concentrations above 1000ng/mL while reducing peak concentrations relative to the dosing protocoldescribed above (400 mg loading dose followed by 200 mg every 12 hoursfor 9 doses) was administration of a 150 mg loading dose followed by 150mg every 6 hours for 7 doses.

REFERENCES

-   1. WHO (2009). “Dengue and dengue hemorrhagic fever fact sheet 117.”    World Health Organization.-   2. Fink J, Gu F and Vasudevan S G (2006). “Role of T cells,    cytokines and antibody in dengue fever and dengue hemorrhagic    fever.” Rev Med Virol 16(4): 263-75.-   3. Halstead S B (2007). “Dengue.” Lancet 370(9599): 1644-52.-   4. Remme J H, Blas E, Chitsulo L et al. (2002). “Strategic emphases    for tropical diseases research: a TDR perspective.” Trends Parasitol    18(10): 421-6.-   5. Courageot M P, Frenkiel M P, Dos Santos C D et al. (2000).    “Alpha-glucosidase inhibitors reduce dengue virus production by    affecting the initial steps of virion morphogenesis in the    endoplasmic reticulum.” Journal of Virology 74(1): 564-72.-   6. Rathore A P S, Paradkar P N, Watanabe S et al. (2011).    “Celgosivir treatment misfolds dengue virus NS1 protein, induces    cellular pro-survival genes and protects against lethal challenge    mouse model.” ANTIVIRAL RESEARCH 92(3): 453-60.-   7. Ng C Y, Gu F, Phong W Y et al. (2007). “Construction and    characterization of a stable subgenomic dengue virus type 2 replicon    system for antiviral compound and siRNA testing.” Antiviral Res    76(3): 222-31.-   8. Schul W, Liu W, Xu H-Y et al. (2007). “A dengue fever viremia    model in mice shows reduction in viral replication and suppression    of the inflammatory response after treatment With antiviral drugs.”    J INFECT DIS 195(5): 665-74.-   9. Zellweger R M, Prestwood T R and Shresta S (2010). “Enhanced    infection of liver sinusoidal endothelial cells in a mouse model of    antibody-induced severe dengue disease/” Cell Host Microbe 7(2):    128-39.-   10. Durantel D (2009). “Celgosivir, an alpha-glucosidase I inhibitor    for the potential treatment of HCV infection.” Curr Opin Investig    Drugs 10(8): 860-70.-   11. Erfle D, Rubinchik E, Pasetka H et al. (2005). “Pharmacokinetics    of celgosivir (MX-3253), a novel a-glucosidase I inhibitor in    loperamide-treated and diarrhea-induced rats.” Antiviral Research    65: 1-1.-   12. Sorbera L A, Castaner J and Garcia-Capdevila L (2005),    “Celgosivir.” Drugs of the Future 30(6): 545-552.-   13. Roth McPherson M, Hamedani P et al. (1996). “Phase 1 tolerance    and pharmacokinetics of a new castanospermine derivative, MDL    28,574A.” Int Conf AIDS Abstracts: 1-1.-   14. Saul R, Ghidoni J J, Molyneux R J et al. (1985).    “Castanospermine inhibits alpha-glucosidase activities and alters    glycogen distribution in animals.” Proc Natl Acad Sci. USA 82(1):    93-7.-   15. Stoltz M and Arumugham T (1996). “Single and multiple dose    proportionality study of MDL 28,574, Study No. NDPRO005.” Hoeschst    Marion Roussel (Report No. K-96-0262-D): 579p.-   16. Beal S L, Sheiner L B, Boeckmann A J, Bauer R J. “NONMEM Users    Guides.” ICON Development Solutions, Ellicott City, Md., USA.    1989-2011.-   17. R. Core Team. R: “A Language and Environment for Statistical    Computing.” R Foundation for Statistical Computing, Vienna, Austria,    2013.-   18. L. Sorbera, Castaner and L. Garcia-Capdevila, “Celgosivir,”    Drugs of the Future, vol. 30, no. 6, pp. 545-552, 2005.-   19. C. Sung, S. Vasudevan, J. Low and E. Ooi, “Celgosivir    Investigator's Brochure,” Duke-NUS Graduate Medical School,    Singapore, 2012.-   20. Duke-NUS Graduate Medical School Program for Emerging Infectious    Diseases, “Clinical Protocol EID-DF-01 Celgosivir Proof of Concept    Trial for Treatment of Acute Dengue Fever (CELADEN),” 2012.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating a dengue virus (DENV) infection in a humansubject, comprising: a) administering to the human subject an initialdose of about 100 to about 600 mg of a compound of Formula (I), or apharmaceutical composition comprising a compound of Formula (I), withinfrom about onset of fever to about 72 hours of fever onset due to dengueinfection; and b) administering to the human subject a dose of about 25to about 300 mg of a compound of Formula (I), or a pharmaceuticalcomposition comprising a compound of Formula (I), at intervals of fromabout 6 to about 12 hours until there is an improvement in the infectionor between from about 1 day to about 10 days, Formula (I) having thefollowing structure,

or a pharmaceutically acceptable salt thereof; wherein R¹, R², and R³are independently H, (C1-C14) acyl, (C1-C14) alkenylacyl, (C3-C8)cycloalkylacyl, (C1-C14) haloalkylacyl (C1-C8) alkoxyacyl, or (C6-C10)arylacyl.
 2. (canceled)
 3. The method of claim 1, wherein the compoundof Formula (I), is a compound of Formula (II):

or a pharmaceutically acceptable salt thereof. 4.-7. (canceled)
 8. Themethod of claim 3, wherein the compound of Formula (II) is converted tocastanospermine after administration to a human subject.
 9. The methodof claim 1, wherein a steady state Cmin serum or plasma concentration ofbetween about 0.05 and about 2.0 microgram/mL of castanospermine in anadult or child human subject is attained after treatment. 10.-13.(canceled)
 14. The method of claim 1, wherein dengue viral infectioncomprises at least one dengue virus selected from DENV1, DENV2, DENV3and DENV4.
 15. The method of claim 1, wherein the dengue viral infectionis secondary dengue infection.
 16. The method of claim 1, wherein thehuman subject to be treated is positive prior to treatment for a dengueinfection using a NS1 strip assay or quantitative PCR.
 17. The method ofclaim 1, wherein the virological log reduction in treated human subjectsis at least 50% greater than untreated or placebo-treated groups. 18.The method of claim 1, wherein administering the compound, or thepharmaceutical composition, achieves a steady state Cmin serum or plasmaconcentration of between about 0.05 and about 2.0 microgram/mL ofcastanospermine. 19.-22. (canceled)
 23. The method of claim 1, whereinthe compound, or the pharmaceutical composition, is administeredintravenously, orally, rectally, or sublingually.
 24. The method ofclaim 1, wherein the human subject is administered an initial dose of150 mg and a dose of 100 mg is thereafter administered to the humansubject every 6 hours, 8 hours, or 12 hours for about 1 day, about 2days, or about 5 days.
 25. The method of claim 1, wherein the humansubject is administered an initial dose of 150 mg and a dose of 150 mgis thereafter administered to the human subject every 6 hours for about1 day, about 2 days, or about 5 days.
 26. The method of claim 1, whereinthe human subject is administered an initial dose of 150 mg and a doseof 150 mg is thereafter administered to the human subject every 8 hoursfor about 1 day, about 2 days, or about 5 days.
 27. The method of claim1, wherein the human subject is administered an initial dose of 150 mgand a dose of 150 mg is thereafter administered to the human subjectevery 12 hours for about 1 day, about 2 days, or about 5 days.
 28. Themethod of claim 1, wherein the human subject is administered an initialdose of 150 mg and a dose of 200 mg is thereafter administered to thehuman subject every 6 hours, 8 hours, or 12 hours for about 1 day, about2 days, or about 5 days.
 29. The method of claim 1, wherein the humansubject is administered an initial dose of 200 mg and a dose of 100 mgis thereafter administered to the human subject every 6 hours, 8 hours,or 12 hours for about 1 day, about 2 days, or about 5 days.
 30. Themethod of claim 1, wherein the human subject is administered an initialdose of 200 mg and a dose of 150 mg is thereafter administered to thehuman subject every 6 hours, 8 hours, or 12 hours for about 1 day, about2 days, or about 5 days.
 31. The method of claim 1, wherein the humansubject is administered an initial dose of 200 mg and a dose of 200 mgis thereafter administered to the human subject every 6 hours, 8 hours,or 12 hours for about 1 day, about 2 days, or about 5 days.
 32. Themethod of claim 1, wherein the human subject is administered a single ora divided dose of about 25 to about 300 mg of the compound or thepharmaceutical composition, for about between about 5 days to about 10days.
 33. The method of claim 32, wherein the human subject isadministered a divided dose of about 25 to about 300 mg of the compound,or the pharmaceutical composition, for about between about 5 days toabout 10 days. 34.-66. (canceled)